Many valuable biotherapeutics in the biotechnology industry are glycoprotein products secreted from mammalian cells. These secreted glycoproteins often undergo post-translational modifications including Nlinked glycosylation (N-glycosylation), which involves the en bloc transfer of an oligosaccharide from a longchain isoprenoid lipid (dolichol) onto a nascent polypeptide containing the consensus sequence Asn-XSer/Thr via a multi-subunit enzyme called oligosaccharide transferase (OST). These oligosaccharide attachments (N-glycans) can be critical to protein properties including folding, stability, resistance to proteases, bioactivity, and in vivo clearance rates. The generation of incompletely N-glycosylated proteins products at positions normally glycosylated indicates a deficiency in either the levels of the dolichol-linked oligosaccharide (DLO) substrate or the OST enzyme that transfers the oligosaccharide onto the target polypeptide. The inability to attain proper N-glycosylation lowers product quality and bioactivity, increases production costs, and inhibits product commercialization. The failure to obtain proper N-glycosylation is also linked to a collection of diseases known as Congenital Disorders of Glycosylation (CDGs) with patients suffering from neural dysfunction, organ failure, and growth retardation. The objective of the current proposed project is to apply metabolic engineering strategies to overcome bottlenecks in the N-glycosylation pathway that lead to the formation of underglycosylated proteins in mammalian cells of biotechnology and biomedical interest. The initial objective will be to examine the intermediates in the DLO metabolic pathway in order to determine which step(s) are causing N-glycosylation deficiency. Once the bottleneck enzyme is elucidated, the next goal will be to engineer relevant mammalian cell lines to overexpress the limiting enzyme or enzymes and increase the efficiency of N-glycosylation of proteins in wild type and mutant mammalian cells. Applying metabolic engineering strategies will provide insights into the metabolic steps controlling N-glycosylation that will be important to improving bioprocesses, treating CDGs disease, and perhaps characterizing the effects of alcoholism. A better understanding of N-linked glycosylation will ultimately lower health care production costs for life-saving therapeutics and provide new insights into ways to treat patients suffering from N-glycosylation disorders. [unreadable] [unreadable] [unreadable]